Methods for identifying agonists and antagonists for human neuronal nicotinic acetylcholine receptors

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid wherein the at least one subunit is an α2 subunit;
• and
• b) measuring ion flux, the electrophysiological response of the cells, or binding of the test compound to the nNAChR, whereby compounds that are agonists or antagonists of the nNAChR are identified, wherein said α2 subunit comprises the sequence of amino acids set forth in SEQ ID NO:2.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α3 subunit;
• and
• b) measuring ion flux, the electrophysiological response of the cells, or interaction of the test compound with the nNAChRs, whereby compounds that are agonists or antagonists of the nNAChR are identified, wherein:
• the α3 subunit comprises a sequence of amino acids encoded by the α3-encoding nucleic acid that is isolated from a plasmid having all of the identifying characteristics of HnAChRα3 deposited under ATCC Accession No. 68278;
• or
• comprises the sequence of amino acids set forth in SEQ ID NO:4.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• b) measuring ion flux, the electrophysiological response of the cells, or interaction of the test compound with the nNAChRs, whereby compounds that are agonists or antagonists of the nNAChR are identified, wherein:
• the α4 subunit is encoded by nucleic acid that hybridizes along its full length under conditions of high stringency to nucleic acid that encodes the sequence of amino acids set forth in SEQ ID No:6 or
• the α4 subunit comprises the sequence of amino acids encoded by α4 subunit encoding nucleic acid in clone HnAChR α4.2, deposited under ATCC Accession No. 69239;
• or
• the α4 subunit comprises the sequence of amino acids encoded by α4 subunit encoding nucleic acid in clone HnAChR α4.1, deposited under ATCC Accession No. 69152;
• the α4 subunit is encoded by nucleic acid that hybridizes under conditions of high stringency to nucleotides 184-2067 of SEQ ID No. 5;
• or
• the α4 subunit is encoded by nucleic acid that hybridizes under conditions of high stringency to the entire α4 encoding insert of clone HnAChR α4.2deposited under ATCC Accession No. 69239.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α6 subunit;
• and
• b) measuring ion flux, the electrophysiological response of the cells or interaction of the test compound with the nNAChRs, whereby compounds that are agonists or antagonists of the nNAChR are identified, wherein:
• the α6 subunit comprises a sequence of amino acids encoded by nucleic acid that hybridizes along its full length under conditions of high stringency to the coding sequence in the sequence of nucleotides set forth in SEQ ID No. 9,
• or the α6 subunit comprises the sequence of amino acids sequence set forth in SEQ ID No. 10.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nHAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α7 subunit and
• b) measuring ion flux, the electrophysiological response of the cells, or interaction of the test compound with the nNAChRs, whereby compounds that are agonists or antagonists of the nNAChR are identified, wherein:
• the α7 subunit comprises a sequence of amino acids encoded by nucleic acid that hybridizes along its full length under conditions of high stringency to the coding sequence in the sequence of nucleotides set forth in SEQ ID No. 11,
• or the α7 subunit comprises the sequence of amino acid sequence set forth in SEQ ID No. 12.

A method for identifying compound that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α5 subunit;
• and
• b) measuring ion flux, the electrophysiological response of the cells, or binding of the test compound to the nNAChR, whereby compounds that are agonists or antagonists of the nNAChR are identified, wherein:
• the α5 subunit comprises a sequence of amino acids encoded by nucleic acid that hybridizes along its full length under conditions of high stringency to the coding sequence in the sequence of nucleotides set forth in SEQ ID No. 7,
• or the α5 subunit comprises the sequence of amino acid sequence set forth in SEQ ID No. 8.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• the recombinant cells express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• the heterologous nucleic acid encodes a β2 subunit of a human nNAChR;
• and
• the expressed nNAChR comprises at least one nNAChR α subunit and the β2 subunit;
• and
• (b) measuring ion flux, the electrophysiological response of the cells, or interaction of the test compound to the nNAChR, whereby compounds that are agonists or antagonists of the nNAChR are identified, wherein the β2 subunit comprises the sequence of amino acids set forth in SEQ ID No. 14.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α3 subunit, wherein:
• the α3 subunit comprises a sequence of amino acids encoded by the α3-encoding nucleic acid that is isolated from a plasmid having all of the identifying characteristics of HnAChR α3 deposited under ATCC Accession No. 68278;
• or comprises the sequence of amino acids set forth in SEQ ID No. 4;
• and
• the recombinant cells further comprise DNA encoding a reporter gene operatively linked to DNA encoding a transcriptional control element, wherein the activity of the transcriptional control element is regulated by the human neuronal nicotinic acetylcholine receptor;
• the reporter gene encodes a detectable gene product, wherein the detectable product is selected from the group consisting of mRNA and a polypeptide;
• and
• b) measuring transcription of the reporter gene by detecting the gene product, whereby compounds that are agonists or antagonists of the nNAChR are identified.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acids is an α4 subunit, wherein:
• the α4 subunit is encoded by nucleic acid that hybridizes along its full length under conditions of high stringency to a nucleic acid that encodes the sequence of amino acids set forth in SEQ ID No:
• 6;
• the α4 subunit is encoded by nucleic acid that hybridizes under conditions of high stringency to the entire α4 encoding insert of clone HnAChRα4.2, deposited under ATCC Accession No. 69239;
• the α4 subunit comprises the sequence of amino acids encoded by α4 subunit encoding nucleic acid in cline HnAChR α4.1, deposited under ATCC Accession No. 69152;
• the α4 subunit is encoded by the nucleic acid that hybridizes along it full length under conditions of high stringency to the entire length of nucleotides 184-2067 of SEQ ID No. 5;
• or
• the α4 subunit is encoded by nucleic acid that hybridizes under conditions of high stringency to the entire α4 encoding insert of clone HnAChRα4.2, deposited under ATCC Accession No. 69239;
• and
• the recombinant cells further comprise DNA encoding a reporter gene operatively linked to DNA encoding a transcriptional control element, wherein the activity of the transcriptional control element is regulated by the human neuronal nicotinic acetylcholine receptor;
• and
• the reporter gene encodes a detectable gene product, wherein the detectable product is selected from the group consisting of mRNA and a polypeptide;
• and
• b) measuring transcription of the reporter gene by detecting the gene product, whereby compounds that are agonists or antagonists of the nNAChR are identified.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α6 subunit, wherein:
• the α6 subunit comprises a sequence of amino acids encoded by nucleic acid that hybridizes along its full length under conditions of high stringency to the coding sequence in the sequence of nucleotides set forth in SEQ ID No. 9,
• or the α6 subunit comprises the sequence of amino acid sequence set forth in SEQ ID No. 10;
• and
• the recombinant cells further comprise DNA encoding a reporter gene operatively linked to DNA encoding a transcriptional control element, wherein the activity of the transcriptional control element is regulated by the human neuronal nicotinic acetylcholine receptor;
• and
• the reporter gene encodes a detectable gene product, wherein the detectable product is selected from the group consisting of mRNA and a polypeptide;
• and
• b) measuring transcription of the reporter gene by detecting the gene product, whereby compounds that are agonists or antagonists of the nNAChR are identified.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α7 subunit, wherein:
• the α7 subunit comprises a sequence of amino acids encoded by nucleic acid that hybridizes along its full length under conditions of high stringency to the coding sequence in the sequence of nucleotides set forth in SEQ ID No. 11,
• or the α7 subunit comprises the sequence of amino acid sequence set forth in SEQ ID No. 12;
• and
• the recombinant cells further comprise DNA encoding a reporter gene operatively linked to DNA encoding a transcriptional control element, wherein the activity of the transcriptional control element is regulated by the human neuronal nicotinic acetylcholine receptor;
• and
• the reporter gene encodes a detectable gene product, wherein the detectable product is selected from the group consisting of mRNA and a polypeptide;
• and
• b) measuring transcription of the reporter gene by detecting the gene product, whereby compounds that are agonists or antagonists of the nNAChR are identified.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α5 subunit, wherein:
• the α5 subunit comprises a sequence of amino acids encoded by a nucleic acid that hybridizes along its full length under conditions of high stringency to the coding sequence in the sequence of nucleotides set forth in SEQ ID No. 7,
• or the α5 subunit comprises the sequence of amino acid sequence set forth in SEQ ID No. 8;
• and
• the recombinant cells further comprise DNA encoding a reporter gene operatively linked to DNA encoding a transcriptional control element, wherein the activity of the transcriptional control element is regulated by the human neuronal nicotinic acetylcholine receptor;
• and
• the reporter gene encodes a detectable gene product, wherein the detectable product is selected from the group consisting of mRNA and a polypeptide;
• and
• b) measuring transcription of the reporter gene by detecting the gene product, whereby compounds that are agonists or antagonists of the nNAChR are identified.

A method for identifying compounds that are antagonists or agonists of human neuronal nicotinic acetylcholine receptors (nNAChRs), said method comprising:

• a) contacting recombinant cells with a test compound, wherein:
• the recombinant cells comprise heterologous nucleic acid encoding at least one human nNAChR subunit;
• express an nNAChR comprising at least one human subunit encoded by the heterologous nucleic acid;
• and
• the at least one human nNAChR subunit encoded by the heterologous nucleic acid is an α2 subunit, wherein:
• the α2 subunit comprises a sequence of amino acids having more than 95% identity with amino acid sequence set forth in SEQ ID No. 2;
• or
• the α2 subunit is encoded by a sequence of nucleotides that hybridizes under conditions of high stringency with the complement of nucleotides 166-1755 set forth in SEQ ID No. 1 or shares at least about 90% identity to nucleotides 166-1755 in SEQ ID No:1;
• or
• α2 subunit comprises the sequence of amino acids set forth in SEQ ID NO:2;
• or
• the α2 subunit is encoded by the nucleotides 166-1755 of SEQ ID No. 1;
• and
• the recombinant cells further comprise DNA encoding a reporter gene operatively linked to DNA encoding a transcriptional control element, wherein the activity of the transcriptional control element is regulated by the human neuronal nicotinic acetylcholine receptor;
• and
• the reporter gene encodes a detectable gene product, wherein the detectable product is selected from the group consisting of mRNA and a polypeptide;
• and
• b) measuring transcription of the reporter gene by detecting the gene product, whereby compounds that are agonists or antagonists of the nNAChR are identified.